Aseptic cell processing and production with no chemical biocides

ABSTRACT

A method and apparatus of aseptic processing and production of cells in a non-sterile enclosure apparatus without chemical biocides is disclosed, by controlling the level of humidity throughout the enclosure to 25% relative humidity (RH) or less, and preferably 20% or 15% or less RH. In addition, the temperature is controlled to 37° C., and consistent gas flow is maintained the enclosure. Colony forming units from microbial contamination detected by environmental monitoring within the enclosure are significantly reduced in this method.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 120 of PCTInternational patent application PCT/US20/56836, filed Oct. 22, 2020,and claims priority to U.S. Patent Application 62/924,322, filed Oct.22, 2019, the entire contents of which is incorporated by reference.

FIELD OF THE INVENTION

This disclosure pertains to a method of processing and production ofcells aseptically in non-sterile apparatus by adjustments of humidity,temperature, and air flow without the use of chemical biocides, andwithout exposing the cells to suboptimal conditions.

BACKGROUND

Cell culture is the process by which cells, typically but notexclusively mammalian cells are grown and handled under controlledconditions outside their natural environment in the body. After thecells of interest have been isolated from living tissue, they cansubsequently be maintained under carefully controlled conditions. Theseconditions vary for each cell type, but generally consist of a suitablevessel with a substrate or medium that supplies the essential nutrients(amino acids, carbohydrates, vitamins, minerals), growth factors,hormones, and gases (CO₂, O₂), and regulation of the physio-chemicalenvironment (pH buffer, osmotic pressure, temperature) at optimum levelsfor those cells.

Cells are used in drug discovery, cancer biology, regenerative medicinedevelopment, and basic life science research, to name a few of manyapplications in research. Industrially cells are also used for vaccineand biologics production, cell therapies, and cell-based gene therapies.

Growing cells ex vivo is technically challenging. To maintain the healthand quality of living cells, the needs of cells must be fully supportedto the extent possible. For example, cells grown ex vivo have no immunesystem to protect them from microbes, so protection against microbialcontamination is required. Cells outside the body no longer have thebody to keep conditions optimal. Temperature, pH, osmolarity, oxygen,carbon dioxide, etc. must be controlled at optimal levels outside thebody or cells will degenerate and die. Conventional equipment onlyprovides part time optimization, only inside incubators or bioreactors.For example, oxygen concentration is a critical parameter for cellprocessing and production. Cells inside the body never see oxygen levelsas high as air oxygen. Physiologic oxygen levels are much lower, andthey do not fluctuate in the body. Air oxygen levels are not physiologicand can damage cells. Accordingly, growing and processing cells ex vivorequires special environmental conditions that must be strictlycontrolled.

In some implementations, cells are grown in specialized isolationchambers specifically adapted to cell processing, manipulation, andproduction applications. For example, such isolation chambers mayinclude a set of modular interconnected chambers, co-chambers, andsub-chambers configured to enclose all steps of a cell productionprocess or series of cell process steps and compartmentalize in order toisolate certain individual steps from adjacent steps. An example of suchequipment is the XVIVO SYSTEM® produced by BioSpherix Ltd, of Parish,New York. The XVIVO SYSTEM® provides a set of modular chambers, boxes,glove boxes, cabinets, sensors, environmental regulation apparatus, andother equipment specifically for cell culture, processing, andproduction applications. Since cells cannot be terminally sterilized,they must be produced by aseptic processing.

Separating cells ex vivo from room air and the people handling themusing a physical barrier such as an isolator, or glove box, or othersimilar type of enclosures, dramatically reduces the chance of microbialcontaminants reaching cells in culture. However, microbes can beentrapped inside upon the initial closing of the enclosure, and canenter a controlled enclosure on the surfaces of materials and suppliesbrought into the enclosure on a routine basis. The use of chemicalbiocides (also termed “microbiocides”) applied as liquid disinfectantsin wipe-downs of internal surfaces of such enclosures, wipe-down ofitems moved into such enclosures, or applied as gaseous fumigationsinside such enclosures is the typical microbial risk mitigationtechnique for creating a sterile or nearly sterile environment inside socells can be aseptically processed and produced. The problem is thatchemical biocides can be toxic and therefore dangerous to people, andmay be toxic to all cells, including the desired cells in culture thatrequire protection from microbial contamination.

SUMMARY OF THE INVENTION

This invention pertains to methods of maintaining a sterile or nearlysterile environment inside a controlled environment enclosure foraseptic cell processing and production without chemical biocides andwithout exposing cells to suboptimal conditions. This method withoutchemical biocides is as effective at enabling aseptic processing andproduction of cells as with chemical biocides, yet non-toxic for cellsbeing processed and safe for personnel operating the cell processingequipment.

Microbes have susceptibilities to temperature (T) and relative humidity(RH). The inventor has found T and RH conditions that reduce andmaintain the microbial bioburden inside enclosures to levels that enableaseptic processing and production of cells without the use of chemicaldisinfectants, without compromising the optimum conditions for the cellsof interest.

In an embodiment, this invention provides a method and environment foraseptic processing and production of cells in non-sterile enclosureapparatus without biocides. The method may employ an enclosure apparatusproviding a controlled environment optimal for ex vivo cultivation,growth, processing or transport of prokaryotic or eukaryotic cells,wherein atmospheric gases, relative humidity (RH), temperature, and gascirculation can be precisely controlled. In the method, the RH of theenclosure is maintained at 25% or less around the clock, except forintervals when higher RH required for steps in the cultivation, growth,processing or transport of cells is temporarily controlled to the lowestRH level necessary and shortest duration necessary only in thecompartment necessary, and then immediately returning the RH to 25% orless. In embodiments, the RH may be maintained at 20% or less, 15% orless, 10% or less, or 5% or less. In an embodiment, the temperature ofthe enclosure may be warmed to about 37° C. to enhance microbial controlyet not be suboptimal for cells. In an embodiment, a continuouslyflowing atmosphere not suboptimal for cells accelerates drying and mixesand homogenizes RH throughout the enclosure.

This method is useful, for example, after initial closure of theisolative apparatus, and after periodic opening and re-closures, whereinall areas and surfaces within the enclosure are rapidly dried toeffectively mitigate microbial contamination risk for cell processingand production operations. The method is also useful when any material,items, or apparatus is moved into the enclosure from the externalenvironment. Such entries are routine in the operation of enclosurechambers. In the method, the materials moved into the enclosure arerapidly dried.

The enclosure apparatus may be a set of interconnected chambers,co-chambers, and sub-chambers, compartmentalized by internal doorsbetween chambers, permanently connected or temporarily connected,monolithic or modular. The enclosure apparatus may be stationary ormobile. The enclosure apparatus may be made of rigid or flexible walls,metal or plastic walls, or any combinations thereof. In an embodiment,the interior surfaces in the contained environment may be made from amaterial selected from polymers, metals, and glass. The interiorsurfaces may be hydrophobic or hydrophilic. The enclosure apparatus maybe fitted with functional or physical inputs/outputs sealed throughwalls, either permanent or temporary. Functional inputs/outputs refersto, for example, wires or tubes between chambers in an enclosureapparatus or to the exterior of an enclosure apparatus. These can beelectrical power conduits, communication wires, gas handling tubes, andthe like.

In an embodiment, the atmospheric gases within the enclosure apparatuscan be precisely controlled, and may be comprised of oxygen, nitrogen,carbon dioxide, nitric oxide, carbon monoxide; and wherein VOC's,particulates, and pressure can be precisely controlled. The atmospherein the contained environment may have an oxygen level at 0.1% to 35%v/v, and carbon dioxide at 0.1% to 20% v/v depending of what is optimalfor cells.

In an embodiment, cells are processed and produced inside the enclosuremanually through sealed ports by gloves or telemanipulators or byintegrated process and analytic machines or full or partial automation.

In an embodiment, the cells grown and processed in the enclosedenvironment are eukaryotic or prokaryotic cells. The eukaryotic cellsmay be mammalian or insect cells or other cells. The cells may be humanor mouse cells or other cells. The cells may be fresh primary cultures,early passage cultures, or cell lines.

In an embodiment, after rapid drying, no measurable CFU's (colonyforming units) are detected floating as measured by environmentalmonitoring comprising settle plates or active air sampling plates.Floating or suspended in the moving atmosphere is the dominant path forcontamination of cells. The only CFU's detectable are strongly adheredto surfaces, as measured by contact plates, where they are sequesteredaway from the cells and statistically unlikely to contaminate cells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the log reduction from the Example of coupons (i.e., testsurfaces) of polypropylene or stainless steel inoculated withPseudomonas aeruginosa, and at various time intervals after exposure tothe inventive conditions of 15% relative humidity (RH) and 37° C. in acontrolled environment enclosure (an XVIVO SYSTEM chamber) or aconventional biological safety cabinet (BSC) at 40% RH and 21° C.Asterisks (*) denote significant within-group differences (compared totime 0) while pound (#) demonstrates significant between-groupdifferences (different conditions) determined by two-way ANOVA followedby Bonferroni's multiple comparisons test (* or #, p<0.05; ** or ##,p<0.01; *** or ###, p<0.001; ****, p<0.0001).

FIG. 2 shows the log reduction of coupons inoculated with Candidaalbicans, a pathogenic yeast, under the same conditions as discussed forFIG. 1.

FIG. 3 shows the log reduction of coupons inoculated with Staphylococcusaureus, a Gram-positive, round-shaped bacterium that is a usual memberof the microbiota of the body, but is implicated in a number ofpathogenic illnesses, under the same conditions as discussed for FIG. 1.

FIG. 4 shows the log reduction of coupons inoculated with Aspergillusbrasiliensis, a fungus ubiquitous in soil that is a common foodcontaminant, under the same conditions as discussed for FIG. 1.

FIG. 5 shows the log reduction of coupons inoculated with Bacillussubtilis, a Gram-positive, catalase-positive bacterium, found in soiland the gastrointestinal tract of ruminants and humans, under the sameconditions as discussed for FIG. 1. This bacteria can form a tough,protective endospore, allowing it to tolerate extreme environmentalconditions.

FIG. 6 summarizes the log reductions in CFU's for the organisms studiedin the Example on polypropylene and stainless steel test surfaces.

FIG. 7 shows the log reduction of S. aureas inoculated on polypropyleneor stainless steel coupons at various relative humidity (RH) levels and37° C. in a controlled environment enclosure. Asterisks (*) denotesignificant between-group differences between 37° C./5% RH group and 21°C./40% RH group determined by two-way ANOVA followed by Sidak multiplecomparisons test (*, p<0.05).

FIG. 8 shows the log reduction of S. aureas inoculated on polypropyleneor stainless steel coupons at various temperature levels and 15% RH in acontrolled environment enclosure. Asterisks (*) denote significantwithin-group differences determined by two-way ANOVA followed by Sidak'smultiple comparisons test (*, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001).

FIG. 9 is a photo of the experiment detailed in Example 2, showing a PBIAir Sampler adjacent to a centrifuge tube with a contaminated cap totest for airborne B. subtilis from the dried cap.

FIG. 10 is an alternative view of the same experiment as shown in FIG.9. This view shows the faceplate/air intake on the Air Sampler device.

DETAILED DESCRIPTION

This invention provides an environment for aseptically processing andproducing cells optimized for cellular existence outside the body(except frozen), particularly in multi-compartment isolative chambersand enclosures. The term “cellular existence” refers to all of the onlyfour states of existence of cells outside the body:

1. Cells proliferating or not proliferating in a state of incubation;

2. Cells being handled or manipulated;

3. Cells being processed or analyzed in some kind of machine;

4. Transport of cells between any of the above three states.

The environments used for such cellular manipulations may be in anenclosure apparatus that may comprise a set of interconnected chambers,co-chambers, and sub-chambers, compartmentalized by internal doorsbetween chambers, permanently connected or temporarily connected,monolithic or modular. The enclosure apparatus may be fixed or mobile.

In the inventive method, rapid extreme drying is the primary physicalmechanism used to create and maintain an aseptic or nearly asepticenvironment desirable for cells. Extreme drying can kill most microbesand prevent the growth of all others. A small proportion of microbes canbe resistant to desiccation. If none of these end up inside theenclosure, this method maintains aseptic conditions. If some desiccationresistant microbes end up inside, this method maintains nearly asepticconditions, yet still enables aseptic cell processing due toimmobilization of the viable microbes on surfaces, thereby sequesteringthem away from cells. It is accomplished by controlling the level ofhumidity throughout the entire system, to an extremely low level, lessthan 25% relative humidity (RH), and preferably 20% or 15% or less RH.In addition, continuous pervasive temperature control throughout at 37°C. enhances the microbicidal effectiveness of desiccating humiditylevels at these low RH levels. In addition, a moving internal atmosphereaccelerates drying and homogenizes antimicrobial conditions throughoutenclosure. Optionally, variable controlled oxygen and variablecontrolled carbon dioxide necessary for optimizing conditions for cellsdoes not interfere with this disinfection protocol. As used herein, theterm “microbe” refers to any undesired contaminating organism, forexample undesirable bacteria or fungi that can contaminate cellcultures.

Whenever high humidity levels are required for a step in a processpertaining to cellular existence, the RH is strictly controlled atminimally necessary levels only in the compartment necessary for onlythe minimum time necessary, and then immediately returned to desiccatinglevels. Whenever lower temperatures are required, they are strictlycontrolled at no lower than necessary only in the compartment necessaryfor only the time necessary, and then immediately returned to 37° C.Temperatures higher than 37° C. enhance microbial kill, but are notnecessary. Data shows this method tips the balance successfully to aradical reduction in contamination risk simultaneously with a radicalreduction in use of risky chemical biocides while not exposing cells tosuboptimal conditions.

In an embodiment, the RH, temperature, and flowing atmosphere may becontrolled for each chamber independently of any other chamber in theenclosure apparatus. For example, a cell optimization protocol requiringa lower temperature or higher humidity could be performed in oneparticular chamber in a multi-chamber apparatus, while other chambersmaintain the inventive conditions of low RH and elevated temperature. Inan embodiment, the environment of each chamber in a multichamberenclosure apparatus can be controlled independently of any otherchamber.

Significantly, no chemical biocides (liquids or gases) are used in theinventive method. Examples of liquid chemical biocides include isopropylalcohol, quaternary ammonium salts, bleach, etc. Examples of gaseouschemical biocides include vaporized hydrogen peroxide, chlorine dioxide,formaldehyde, etc. None are necessary. The inventor has discovered thatlow humidity, elevated temperature, and turbulent or laminar gas flowcan sufficiently sanitize internal environment including the atmosphereand surfaces in enclosures and chambers optimized for cells.

Presumably, the low humidity conditions of this invention kills mostmicrobial organisms because they are sensitive to desiccation, and fororganisms capable of surviving low humidity and elevated temperatures,for example from spore forming bacteria and fungi, all microbesincluding such organisms were discovered to be immobilized on internalsurfaces by strong adhesion to such surfaces. Furthermore, the gas flowused in the inventive method accelerates microbial drying and ensuresthat the desiccating and warm conditions penetrate to all corners andrecesses within the interior of an enclosure apparatus. Spores or otherpotentially infectious particles immobilized on surfaces by rapid dryingunder desiccating conditions are not a measurable contamination risk forcells processed and produced in the enclosures used in this invention.

The inventive method does not require chemical sterilization of thechamber or enclosure in advance, does not require a disinfectantwipe-down of inside surfaces in any part of the system, does not requirea disinfectant wipe down of any materials or equipment moved into theenclosure, and does not require internal washing to reliably process andproduce cells aseptically. The inventive method disinfects and cleansthe interior of an enclosure apparatus sufficiently so that no colonyforming units (CFUs) can be detected floating inside as evidenced byintensive environmental monitoring with settle plates and active airsampling plates. With this invention, over time, an enclosure becomesprogressively more aseptic. However, the inventive method is notincompatible with the use of chemical biocides and may be synergisticwith cautious biocide treatment that does not endanger the cells.

Contact plates (also termed “touch plates”) are used for monitoringmicrobial contamination of surfaces. These plates have an agar mediumpoured into petri dish, and the agar can be contacted with a surface ina chamber. Any microbial contamination on the test surface will adhereto the agar. The agar is then incubated and the microbial contaminationwill grow, which are termed “colony forming units” (CFU's), which can becounted to quantify the degree of contamination on the test surface.Settle plates are similar and are used for passive air monitoring. Anagar plate in a petri is exposed to an environment for a measured lengthof time. Airborne microbial particles will land on the agar. The plateis then incubated and CFU's can be counted. Active air sampling platesemploy an air sampler to physically draw a pre-determined volume of airand pass it over the agar. The plate is then removed from the airsampler and directly incubated. These were used to develop the method.

In this invention, no CFUs are detected by any of these three methods ifno desiccation resistant microbes happen to be inside, wherein theinventive method actually sterilizes the enclosure and makes theenclosure aseptic. However, if any desiccation resistant microbes happento be inside, no CFUs will be detected in only settle plates and activesample plates because immobilization assures whatever few desiccationresistant microbes might be in a chamber subject to the inventivephysical conditions, they are prevented from floating by adhesion to asurface. Such desiccation resistant microbes may be viable and could bedetected by contact plates. In this case the inventive method doesn'tcreate an aseptic environment, or aseptic conditions inside theenclosure, or a sterile enclosure, because it is not sterile inside,only nearly sterile, or nearly aseptic. Inventively, however, it doesenable aseptic processing because the only microbes viable and capableof contaminating the cells are sequestered to a surface. They cantransfer to other surfaces by touch but they don't detach from theseother surfaces. Therefore, there is no path to contaminate cells becausethe practice of sterile technique assures that these few microbes willhave no sequential touch points to any surfaces that will touch thecells or substrates of the cells.

The inventive method may be used, for example, after the initialinstallation and closure of an enclosure apparatus, and after periodicopening and re-closures. Such an apparatus may comprise a set ofinterconnected chambers, co-chambers, and sub-chambers,compartmentalized by internal doors between chambers, permanentlyconnected or temporarily connected, monolithic or modular. The enclosureapparatus may be fixed or mobile. The enclosure apparatus may be made ofrigid or flexible walls, metal or plastic walls, or any combinationsthereof.

The inventive method may also be also use for frequent operations wherematerials, items, and equipment are routinely moved into an enclosureapparatus. Such routine operations are typically the single source ofnew microbial contamination risk. But with the rapidly desiccatingconditions as provided herein, microbes on these materials and equipmentare immobilized within minutes, and most are killed within hours.

The traditional reliance on frequent application and over-use of strongliquid and gaseous chemical biocides to achieve aseptic processing andproduction is highly risky for the desired cells. Furthermore, biocideuse is inherently intermittent. The biocide (liquid or gas) is appliedand then stopped. In between, there is no antimicrobial activity,providing a window for undesirable microbes to grow and contaminateequipment and cell cultures within the enclosure. By contrast, thisphysical approach is constant, with continuous antimicrobial activitymaintained 24/7.

Furthermore, unlike liquid biocide effectiveness limited by surfacecoverage, this physical approach acts like a gas. It reaches into everynook and cranny inside the entire system, especially when driven by gasflow patterns inside the chambers. All interior surfaces in a chamber,whether reachable or unreachable, including inside every crack, seam,crevice, and cavity are permeated with this antimicrobial actioncontinually. Finally, instead of concern with surface residuals andoff-gases and toxic vapors left after each chemical biocide application,this alternative approach leaves no residuals or any toxic off-gases orvapors because it's a physical approach—desiccation at temperatureselevated above room air conditions accelerated by a moving atmosphere.

In an embodiment, gas flow within an enclosure apparatus may be animportant feature in this invention. Gas flow can be turbulent orlaminar. Gas flow relies on a fan that recirculates the controlledatmosphere within each isolated chamber to create some degree ofturbulence within the chamber. In an embodiment, the gases beingrecirculated may also pass through a HEPA filter. Alternatively, gasflow may be laminar, meaning a steady flow in a single direction.

The RH levels, temperature, and gas flow in this invention may becontrolled as other environmental parameters in an isolated chamberapparatus are controlled. For example, RH can be controlled using drygases from gas tanks, which are supplied in a highly purified state withno moisture. RH can also be controlled in established atmospheres usinga recirculation fan that passes the gases in an enclosure overregenerable chemical desiccants (for example, silica gel or calciumsulfate) and back into the enclosure. RH can also be controlled byelectronic or compressor dehumidifiers. In addition, a HEPA filter maybe used in such a recirculation system.

In an embodiment, the drying effect as described herein may be rapid,meaning that when an object is moved into a chamber from an externalenvironment, the humidity and any surface moisture on the object isdried to the point that contaminating microbes are immobilized withinminutes and killed within hours, to achieve the killing or adhesion ofmicrobial contaminants as disclosed herein. This rapid drying minimizesthe ability of contaminants to become airborne within the chambereliminating the major path whereby microbes can contaminate cells.

Microbes can be entrapped inside an enclosure apparatus during assemblyand installation, and can be entrapped after periodic opening andre-closure of part of a system or entire system. With this method thecontamination risk they present drops continuously over time since nonecan reproduce, and most are killed by desiccation. However, a smallpercentage of microbes entrapped may be desiccation resistant. Theirincidence is likely to be different at different sites, and likely tovary at each location over the seasons. Any that become detached andfloat immediately get removed from the processing area and sequesteredpermanently in a remote filter, thereby eliminating them as a risk tothe cells. The few that might remain attached to an internal surface arenot a measurable risk for the same reason, because they get sequesteredto that surface. Under normal operating conditions, the incidence ofthese residual sequestered viable microbes is so small that within a fewdays no CFUs can be detected inside by intensive environmentalmonitoring, not only with settle plates and active air sampling plates,but contact plates as well.

Thereafter the only new bioburden risk comes from surfaces of materialsbrought into the system. Bioburden here is defined as the number ofbacteria living on a surface of the incoming items. Risk is highest nearthe entry point but drops precipitously to undetectable levels along thefirst few sequential points of contact with those materials as they aremoved in. Rapid drying sufficiently mitigates all detectable floatingCFU risk, including desiccation resistant microbes. No gas or liquidchemical biocides are required to routinely produce cells aseptically.

In an embodiment, the atmosphere in the contained environment having anoxygen level at 0.1% to 35% v/v, and carbon dioxide at 0.1% to 20% v/v,with the balance nitrogen. Other gases possibly employed in cellularprocessing and production protocols in the contained environmentsdescribed in this invention may include nitric oxide and carbonmonoxide. Other atmospheric features that can be controlled are volatileorganic compounds (VOC's) which may be introduced from biocidematerials, particulates in the atmosphere of a chamber, and atmosphericpressure.

In an embodiment, the interior surfaces in the chamber with thecontrolled environment are hydrophobic or hydrophilic. In an embodiment,the interior surfaces in the contained environment may be made frompolypropylene or stainless steel. Additional materials are within thescope of this invention, including polyethylene or other rigid plastics,glass, aluminum, and other polished or painted metallic materials.

In an embodiment, the cells processed and produced within the controlledenvironment are eukaryotic cells, which includes mammalian cells, forexample, freshly biopsied primary cultures, or early passage culturesfrom various tissue, or cell lines such as GH3 (rat pituitary tumor) andPC12 (rat pheochromocytoma). In an embodiment, the eukaryotic cells inthe chamber with the controlled environment are human cells, forexample, freshly biopsied primary cultures, early passage cultures, orcell lines MCF-7 (breast cancer), MDA-MB-468 (breast cancer), PC3(prostate cancer), and SaOS-2 (bone cancer) (representative examplesonly). In an embodiment, the cells may be plant cells, or insect cells,or prokaryote cells.

Example 1

Organisms and Media. The following organisms were tested in theinventive method: Pseudomonas aeruginosa, Staphylococcus aureus,Bacillus subtilis, Aspergillus brasiliensis and Candida albicans fromBIOBALL® (BIOMERIEUX (Hazelwood, Mo.). CFU were assayed on cultureplates containing Tryptic Soy Agar from Sigma (St. Louis, Mo.). A.brasiliensis plates were cultured at 25° C. for 36-48 hrs while theother organisms were cultured at 35° C. for 20-24 hrs before colonyassessment. For environmental monitoring, contact plates were made ofBBL™ Trypticase™ Soy Agar from BD (Sparks, Md.). Contact plates wereincubated at 35° C. for at least 20-24 hrs.

Coupon Inoculation. Coupons (10 mm diameter) were made of polypropylene,or stainless steel (Beadthoven Jewelry on Amazon.com). The coupons weretriple-cleaned/disinfected, in TexQ (Texwipe, www.texwipe.com), thenSporKlenz (Steris, Inc. www.steris.com), then 70% ethanol for 30-60 mineach soak, with a triple ddH₂O rinse between each disinfectant. Theywere air dried in a laminar flow hood. Dried coupons were stored insterile 50 ml conical tubes (CELLTREAT Scientific Products; Pepperell,Mass.) at RT.

Microbial Reduction Assays. At least one day prior to these studies, theprobable risk surfaces in the chamber were disinfected with SporKlenz(Steris, Inc. www.steris.com) and atmospheric gases were replaced withfresh triple-filtered dry tanked gases (20%02, 0.1% CO₂, balance N₂) toeliminate any disinfectant fumes. Triple-cleaned/disinfected couponswere inoculated in place on the PC floor as if a drop of bacterialculture had contaminated the work surface. Inoculated coupons wereexposed to experimental conditions and collected at time intervals.Harvested coupons were placed in 1 ml 0.05% Tween-80 in DPBS andvortexed 5×10 seconds. Microbial suspensions were diluted further beforebeing spread on agar plates. Colonies on each plate were counted by twoindividuals who were blinded to experimental conditions. At 10% orgreater discrepancy, a third person re-counted colonies. The mean of twocloser numbers was used for data analysis. Log reduction was calculatedusing equation: R_(f)=log (Y₀)−log (Y_(i)). R_(i) is log reduction foreach time point, where Y₀ is remaining microbes at time zero, and Y_(i)is remaining microbes at time i. All statistical analyses were performedusing GraphPad Prism (Version 8.4.2, GraphPad Software, Inc.) asdescribed in figure legends. Data are expressed as the mean+SEM.Significance was assessed at p<0.05.

The inventive conditions produce larger microbial reductions than roomair BSC conditions in a microbe-dependent manner. The experimentalhypothesis was that there would be differences between microbialinfectivity in controlled enclosure conditions and conventional room airbiological safety cabinet (BSC) conditions. The coupons described above(polypropylene or stainless steel) were inoculated with known number ofmicrobes in each chamber and incubated either in an XVIVO Systemprocessing chamber under inventive conditions (37° C./15% RH), or aprocessing chamber set to conventional room air BSC conditions (21°C./40% RH). Coupons were collected at intervals and assayed forremaining viable colony-forming units (CFU). Data from 3 or moreindependent experiments were combined for comparisons. Statisticallysignificant log reductions in CFU were found on four out of fivemicrobes in the two different conditions. These reductions were onlyfrom exposure to physical atmospheric conditions without the use of anyantimicrobial chemicals.

Data showing differences in CFU recovered over time in each condition,as well as differences between conditions at various time points areshown in FIGS. 1-5 for both polypropylene and stainless steel testsurfaces. P. aeruginosa (FIG. 1), C. albicans (FIG. 2), and S. aureus(FIG. 3) on both materials all demonstrated dramatic and significant logreductions in CFU's within the time frames studied under both inventiveand room air BSC conditions. Results for A. brasiliensis (FIG. 4) wereless dramatic but still significant. Consistent with its knownresistance to environmental conditions, little CFU reduction was seen inB. subtilis spores (FIG. 5) in any condition tested. Comparing themicrobial response to each condition separately (FIG. 6) the dataclearly demonstrated a spectrum of microbial sensitivities to cellprocessing chamber conditions which were consistent with their knownsensitivities to environmental conditions. These trends were similarbetween the two surface materials tested. Asterisks in FIG. 1-4 (*)denote significant within-group differences (compared to time 0) whilepound (#) demonstrates significant between-group differences (differentconditions) determined by two-way ANOVA followed by Bonferroni'smultiple comparisons test (* or #, p<0.05; ** or ##, p<0.01; *** or ###,p<0.001; ****, p<0.0001).

Various humidity levels in the controlled environmental chamber werealso investigated (FIG. 7), from between 40% RH to 5% RH. Significantincreases in log reductions of CFU's are clearly seen in bothpolypropylene and stainless steel as humidity decreases.

Various temperature levels were also investigated as shown in FIG. 6 forS. aureas. This data shows that increasing the temperature from 21° C.to 37° C. resulted in a significant reduction in CFU's detected.

Example 2

Airborne B. subtilis was quantified after rapid drying in the inventivemethod with active air sampling and passive air sampling (settle plates)in XVIVO SYSTEM enclosure.

In an experiment set up as shown in FIGS. 9-10, an air sampling machine3 (“PBI Air Sampler SAS Super 100”) with faceplate/air intake 3 a is setup in enclosure chamber 1 having enclosure floor 2. Centrifuge tube 4having centrifuge tube cap 5 was placed within a few cm's of faceplate 3a just upstream of sample inlet. In addition, several passive airsampling settle plates of agar 6 were also positioned on the floor. Theatmosphere handling arrangement in the enclosure chamber created gasflow turbulence in the chamber.

Cap 5 was contaminated with 10⁷ CFUs of B. subtilis in a tiny drop ofsaline and moved into the chamber with 15% RH at temperature 37° C., andwith a turbulent atmosphere. Immediately after visible drying, whichtook about 11 minutes, the active air sampler with agar plates was usedto quantify any B. subtilis that may have blown off the cap 5.Additional detection of B. subtilis used the array of agar settle plates6 around the air sampler 3. No CFUs were detected over 4 hours in anyplates. The active air sampling was set at 90 L/min and maintained for 4hours with fresh plates every 20 minutes.

This experiment is typical of dozens of other experimental resultsconfirming that rapidly dried microbes on surfaces are immobilizedin-place and despite movement of the atmosphere they do not easilydetach and they do not become airborne, although they will transfer fromsurface to surface upon contact of these surfaces.

The invention claimed is:
 1. A method of aseptic processing andproduction of cells in a nearly aseptic enclosure apparatus withoutbiocides comprising a. at least one chamber in an enclosure apparatushaving a plurality of chambers providing a nearly aseptic environmentadapted for optimal ex vivo cultivation, growth, processing or transportof prokaryotic or eukaryotic cells, wherein atmospheric gases, relativehumidity (RH), temperature, and gas circulation can be preciselycontrolled; b. adjusting the RH of the at least one chamber to 20% orless with a nearly continuously flowing atmosphere optimal for cells tomix and homogenize the RH throughout the at least one chamber; c.adjusting the temperature of the at least one chamber to 37° C. or otherelevated temperature optimal for cells; d. wherein the RH, temperatureand flowing atmosphere within the at least one chamber elicits aphysical antimicrobial effect to disinfect and clean the interior of anenclosure apparatus by rapid desiccation of any microbes floating orsuspended in the atmosphere in the interior of the at least one chamberor on the interior walls or other surfaces of the at least one chamber,resulting in the death or immobilization by adhesion of all microbes onany surfaces in the at least one chamber, whereby microbial reproductionis prevented and no floating or airborne viable undesirable microbes canbe detected; e. wherein any higher RH in the at least one chamberrequired for the cultivation, growth, processing or transport of cellsis controlled to the RH level no higher than necessary for the shortestduration necessary, and then immediately returning the RH to 20% orless; f. wherein any lower temperature in the at least one chamberrequired for the cultivation, growth, processing, or transport of cellsis controlled to the temperature no lower than necessary for theshortest duration necessary, and then immediately returning to theelevated temperature of about 37° C. optimal for cells; and g. wherein(i) upon initial closure and after each periodic opening and re-closureof the enclosure apparatus, all areas, atmosphere, and surfaces withinthe enclosure are rapidly dried; or (ii) any material or apparatus, whenmoved into the enclosure from the external environment is rapidly dried,to create and maintain an aseptic or nearly aseptic environment optimalfor the ex vivo cultivation of cells.
 2. The method of claim 1, whereinthe relative humidity is controlled to 15% or less, or to 10% or less.3. The method of claim 1, wherein the enclosure apparatus comprises twoor more interconnected chambers, co-chambers, and sub-chambers,compartmentalized by internal doors between chambers, permanentlyconnected or temporarily connected, monolithic or modular.
 4. The methodof claim 3, wherein the RH, temperature, and flowing atmosphere iscontrolled for each chamber independently of any other chamber in theenclosure apparatus.
 5. The method of claim 1, wherein the enclosureapparatus is stationary or mobile.
 6. The method of claim 1, wherein theenclosure apparatus is made of rigid or flexible walls, metal or plasticwalls, or any combinations thereof.
 7. The method of claim 1, whereinthe interior surfaces are hydrophobic or hydrophilic.
 8. The method ofclaim 1, further comprising interior surfaces in the containedenvironment made from a material selected from polyethylene,polypropylene, stainless steel, and glass.
 9. The method of claim 1,wherein the enclosure apparatus is fitted with functional or physicalinputs/outputs through walls, either permanent or temporary, eithersealed or semi-sealed in passageways not compromising control insideenclosure.
 10. The method of claim 1, wherein the atmospheric gaseswithin the enclosure apparatus that can be precisely controlled compriseoxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide; andwherein VOC's, particulates, and pressure can be precisely controlled.11. The method of claim 1, further comprising an atmosphere in thecontained environment having an oxygen level at 0.1% to 35% v/v, andcarbon dioxide at 0.1% to 20% v/v.
 12. The method of claim 1, whereincells are processed and produced inside the enclosure manually by glovesor telemanipulators through sealed ports or by integrated process andanalytic machines or by full or partial automation.
 13. The method ofclaim 1, wherein the cells are eukaryotic cells.
 14. The method of claim13 wherein the eukaryotic cells are mammalian cells.
 15. The method ofclaim 14, wherein the mammalian cells are human cells.
 16. The method ofclaim 1, wherein no measurable CFU's (colony forming units) ofdesiccation resistant microbes are detected as measured by environmentalmonitoring with settle plates or active air sampling plates inside theenclosure.
 17. The method of claim 1, wherein no measurable CFU's(colony forming units) of microbes susceptible to desiccation aredetected as measured by environmental monitoring with settle plates,active air sampling plates, or contract plates.